Home About us Contact
|
Home About us Contact | ||
|
|
||
Site-specific Recombination (site-specific + recombination)
Selected AbstractsThe Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructsDEVELOPMENTAL DYNAMICS, Issue 11 2007Kristen M. Kwan Abstract Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter],[coding sequence],[3, tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence,driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088,3099, 2007. © 2007 Wiley-Liss, Inc. [source] Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on rootsENVIRONMENTAL MICROBIOLOGY, Issue 4 2003N. Carol Casavant Summary We constructed and characterized a plasmid-based genetic system that reports the expression of a toluene-responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site-specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80,100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 µm toluene for one to three hours. Compared to the response of A506 containing a plasmid-borne PtbuA1 - gfp fusion, the recombination-based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1 -promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant- or rhizosphere microbial community-derived inducers of the PtbuA1 promoter. This recombination-based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment. [source] Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in RiceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2009Shaohong Qu Abstract Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of AcTPase via Cre- lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element (HPT-Ds). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community. [source] Excision of selectable marker genes from transgenic crops as a concern for environmental biosafetyJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2007Satheesh Natarajan Abstract The main task in the development of transgenic plants is the capability to distinguish between plant cells with an integrated transgene and the bulk of non-transformed cells. Selectable marker genes are required to achieve this goal within the transgene, and to select for their expression. These selectable markers are mostly based on genes conferring antibiotic or herbicide resistance. The presence of the marker gene will lead to unpredictable environmental hazards, so on the basis of economic incentives and safety concerns, several methods, such as site-specific recombination, homologous recombination and co-transformation, have been developed to eliminate these genes from the genome after successful transformation has been achieved. Gene transfer without the incorporation of an antibiotic-resistance marker or herbicide-resistance marker in the host genome should convince the public with regard to the field release of transgenic organisms. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host. Copyright © 2007 Society of Chemical Industry [source] Regulated site-specific recombination of the she pathogenicity island of Shigella flexneriMOLECULAR MICROBIOLOGY, Issue 5 2004Harry Sakellaris Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3, terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase-mediated excision occurs via recombination between a 22 bp sequence at the 3, terminus of pheV and an imperfect direct repeat at the pheV -distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase-mediated integration into the chromosome. The circle junction consists of the pheV -proximal and pheV -distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3, terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase-dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2. [source] Conjugative transposons: the tip of the icebergMOLECULAR MICROBIOLOGY, Issue 3 2002Vincent Burrus Summary Elements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have a diversified set of characteristics including cell-to-cell contact using pili or cell aggregation, transfer of single-stranded or double-stranded DNA, low or high specificity of integration and serine or tyrosine recombinases. This has led to a highly heterogeneous nomenclature, including conjugative transposons, integrative ,plasmids', genomic islands and numerous unclassified elements. However, all these elements excise by site-specific recombination, transfer the resulting circular form by conjugation and integrate by recombination between a specific site of this circular form and a site in the genome of their host. Whereas replication of the circular form probably occurs during conjugation, this replication is not involved in the maintenance of the element. In this review, we show that these elements share very similar characteristics and, therefore, we propose to classify them as integrative and conjugative elements (ICEs). These elements evolve by acquisition or exchanges of modules with various transferable elements including at least ICEs and plasmids. The ICEs are probably widespread among the bacteria. [source] A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants,PLANT BIOTECHNOLOGY JOURNAL, Issue 8 2008Vesna Djukanovic Summary The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-µm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system. [source] Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vectorBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Shuohao Huang Abstract Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717,729. © 2010 Wiley Periodicals, Inc. [source] An accumulative site-specific gene integration system using cre recombinase-mediated cassette exchangeBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2010Yujiro Kameyama Abstract The Cre- loxP system is frequently used for site-specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms. Biotechnol. Bioeng. 2010;105: 1106,1114. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||